Introduction of genes into mammalian cells by transfection leads to their stable integration into the genome of host cells. Usually, this integration event is rare (<0.01%) and occurs in a random way regarding the locus of integration. Expression of the integrated transgene depends on the local environment. This means that nearby enhancers or silencers may affect the expression, and genes may become inactivated by spreading heterochromatin (chromatin position effect, CPE, also called silencing).
During the past years, studies begun in Drosophila and now extended to vertebrates, have identified DNA sequence elements called insulators that appear to function as “barrier”, by preventing CPE, and/or as “enhancer blocking” having the capacity to shield a promoter from the action of a distal enhancer without preventing the enhancer from working on a proximal promoter. Thus, insulators are DNA sequence elements that protect transcribed regions from distant unrelated regulatory sequences.
The first DNA sequences described as having the properties of an insulator were scs (specialized chromosome structures) elements of Drosophila. Chung and Felsenfeld described an element, 5′HS4 (Dnase I-hypersensitive Site), acting as an insulator contained within a 1.2 kb DNA fragment derived from the 5′ end of the chicken B-globin locus (Chung et al., 1993, U.S. Pat. No. 5,610,053). Much of the activity of the insulator was shown to be contained in a 250 bp fragment, the “core” region, that is included in the 5′HS4 sequence (Chung et al., 1997). The DNA of the core region was further dissected into five footprinted regions (FI-FV) and was shown to be GC-rich with the properties of a “CpG island”, but did not appear to function as a promoter. Experiments showed that only one region, FII, was necessary for the enhancer blocking property and the purification of its binding proteins revealed that binding of CTCF (CCCTC-binding factor) was responsible for its activity (Bell et al., 1999).
In another study, the utility of the full 1.2 kb beta globin insulator element in protecting reporters form CPE has been demonstrated in in vivo assays including transgenic mice (Ciana et al., 2001). In CHO cell lines, Izumi and Gilbert showed that the presence of chromatin insulator sequences moderately improved stability but was not sufficient to produce homogeneous transformants (Izumi and Gilbert, 1999).
In a recent publication (Recillas-Targa et al., 2002) the functional activity of a 1.2 kb insulator element from the chicken beta-globin gene was reviewed. Besides working as efficient barrier to the activity of nearby enhancers or silencers, the 250 bp core element was shown to be sufficient to confer protection against silencing of genes caused by CPE and to provide long-term stable expression. Two copies of smaller fragments of the 250 bp core sequence essentially devoid of footprint region II, on each side of the reporter gene, were sufficient to confer protection from CPE, but not enhancer blocking, in chicken pre-erythroid 6C2 cells.
Since vector sizes should preferably not exceed 10 kb, there is a need to reduce the size of the regulatory elements present on these vectors. For instance, vector stability increases as the size of its DNA is reduced. Smaller vectors can accommodate larger segments of inserts. Moreover small elements simplify the modification of expression vectors and allow constructions where the total length of the insert must be limited.
It is therefore an object of this invention to provide a short DNA element that has insulator activity when used in vectors.